Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.     

Possible role of nitric oxide in radiation-induced salivary gland dysfunction

In this study, we developed a murine model of xerostomia to elucidate the mechanism of radiation-induced salivary gland dysfunction and determined the levels of nitric oxide (NO) in the salivary glands to assess its involvement in the salivary dysfunction induced by radiation. In addition, an inhibitor of NO synthesis was administered to the model in vivo, and its effect on saliva secretion was investigated. Salivary gland irradiation at a dose of 15 Gy caused a significant decrease in secretion compared to unirradiated salivary glands. There were no marked differences between the irradiated mice and unirradiated mice in water or food consumption or in body weight changes. The NO levels in the cultured salivary gland epithelial cells were increased by treatment with a combination of interferon gamma (Ifng), interleukin 1-beta (Il1b), and tumor necrosis factor alpha (Tnfa). Irradiation increased the NO level in the salivary gland tissue. The presence of N(G)-monomethyl-l-arginine acetate (l-NMMA), an inhibitor of NO synthesis, caused a decrease in the NO level in cultured salivary gland tissues after irradiation. Administration of l-NMMA to irradiated mice improved saliva secretion. These results suggest that excessive production of NO induced by radiation is involved in the formation of radiation-induced xerostomia. The finding that administration of an inhibitor of NO synthesis ameliorated the dysfunction of irradiated salivary glands indicates that NO plays a role as a mediator of the dry mouth symptoms that occur after irradiation.     

Distribution and properties of arginase in the salivary glands of four species of laboratory mammals

Important progress in arginine metabolism includes the discovery of widespread expression of two isoforms of arginase, arginase I and II, not only in hepatic cells but also in non-hepatic cells, and the formation of nitric oxide, a widely distributed signal-transducing molecule, from arginine by nitric oxide synthase. Possible physiological roles of arginase may therefore include regulation of nitric oxide synthesis through arginine availability for nitric oxide synthase. In this paper, arginase was investigated in the submandibular, sublingual, and parotid glands of rat, mouse, guinea pig, and rabbit. From their arginase contents, the salivary glands of these species were divided into two groups. Variable levels of arginase activity were detected in the salivary glands of mouse and rat. However, salivary glands of rabbit and guinea pig had almost no arginase activity. The presence of nitric oxide synthase has been reported in all the salivary glands used in this study. Therefore, one of the important findings was the presence of species specificity in the co-localization of arginase and nitric oxide synthase in the salivary glands of the four species. The highest specific activity of arginase was found in mouse parotid gland. In rat, considerable arginase activity was detected in all three glands, at 3.6–7.3% of that in rat liver. In rat submandibular gland, arginase was detected in both cytosolic and particulate fractions. In addition, arginase was detected in isolated acinar cells, but not in duct cells. Experiments on the intracellular distribution and the effects of the arginase inhibitors ornithine and N-hydroxy-L-arginine (NOHA), suggested the presence of both arginase I and arginase II in rat submandibular gland.Abbreviations cGMP cyclic guanosine 3,5-monophosphate - NO nitric oxide - NOHA N-hydroxy-L-arginine - NOS nitric oxide synthase Communicated by I.D. Hume     

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.     

IL-17 sequestration via salivary gland gene therapy in a mouse model of Sjogren’s syndrome suppresses disease-associated expression of the putative autoantigen Klk1b22

IntroductionIL-17 has a putative role in the pathophysiology of Sjogren’s syndrome (SS) and has been shown to be upregulated in the salivary glands of affected individuals. Sequestration of IL-17 with Adenoviral-mediated gene therapy has previously shown a benefit upon the SS-like phenotype in the Aec1/Aec2 mouse model. We sought to understand the proteomic consequences of IL-17 sequestration in the salivary gland of this mouse model as a means of illuminating the role of IL-17 in SS-like disease.MethodsUltrasound-assisted gene transfer (UAGT) was utilized to express a fusion protein composed of the extracellular portion of the IL-17 receptor fused to fragment of crystallization (Fc) in the submandibular glands of Aec1/Aec2 mice at 8 weeks of age. After confirming expression of the fusion protein and local and systemic sequestration of IL-17, proteomic profiling was performed on submandibular glands of a treated cohort of Aec1/Aec2 animals relative to the background strain and sham-treated animals.ResultsThe most notable proteomic signatures of IL-17 sequestration on SS-like disease-related proteins were Kallikrein-related peptidases, including the putative autoantigen Klk1b22. IL-17 sequestration also notably led to an isoelectric shift, but not a molecular weight shift, of Kallikrein-1, attributed to phosphorylation.ConclusionNon-viral IL-17 sequestration gene therapy in the salivary gland is feasible and downregulates expression of a putative SS autoantigen in the Aec1/Aec2 mouse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0714-2) contains supplementary material, which is available to authorized users.     

Zymographic demonstration of lactate and malate dehydrogenases isoenzymes in the rodent salivary glands

Summary Analysis of lactate and malate dehydrogenase zymograms of rodent salivary glands showed species and organ specific patterns.Lactate dehydrogenase isoenzyme patterns occupied the middle positions in relation to those of skeletal and heart muscle. Activities of the major salivary glands were in the order submaxillary gland>parotid>sublingual gland. Zymogram of the mouse and rat showed LDH₄ and LDH₅ high activity patterns, while that of the rabbit was the fast moving active one. Hamster salivary gland exhibited a neutral type of the former and the latter.Malate dehydrogenase isoenzyme exhibited very similar patterns for the mouse, rat and hamster. Malate dehydrogenase zymogram of rabbit showed 3 active bands, which was different from the other rodents.     

Metabolic and oxidative impairments in human salivary gland cells line exposed to MeHg

Background/aimThe ingestion of contaminated seafood by MeHg is considered the main route of human exposure, turning the salivary gland one important target organ. The salivary glands play critical roles in maintaining oral health homeostasis, producing saliva that maintains the oral microbiota, initiation of the digestion of macromolecules, and being essential in maintaining the integrity of the adjacent soft tissues and teeth. Thus, this study aimed to investigate the effects of MeHg exposure on human salivary gland cells line.MethodsCells were exposed to 1–6 μM of MeHg for 24 h, and analysis of toxicity was performed. Based on these results, the LC50 was calculated and two concentrations were chosen (0.25 and 2.5 μM MeHg) to evaluate intracellular mercury (Hg) accumulation (THg), metabolic viability and oxidative stress parameters (GSH:GSSG ratio, lipid peroxidation, protein oxidation and DNA damage).ResultsThe results demonstrated accumulation of THg as we increased the MeHg concentrations in the exposure and, the higher the dose, the lower is the cell metabolic response. In addition, the 2.5 μM MeHg concentration also triggered oxidative stress in human salivary gland cells by depleting the antioxidant competence of GSH:GSSG ratio and increasing lipid peroxidation and proteins carbonyl levels, but no damages to DNA integrity.ConclusionIn conclusion, although these two elected doses did not show lethal effects, the highest dose triggered oxidative stress and new questionings about long-term exposure models are raised to investigate furthers cellular damages to human salivary gland cells caused by MeHg exposure to extrapolate in a translational perspective.     

Cytochemical localization of peroxidase activity in the submandibular salivary gland of the hamster

Summary Peroxidase activity has been localized to duct cells of the submandibular salivary gland of the hamster using a 3,3-diaminobenzidine (DAB)-H₂O₂ medium. In cryostat sections of glutaraldehyde-fixed tissue the enzyme activity is found in the proximal part of the duct system of the gland. In Epon sections studied in the light microscope or thin sections studied in the electron microscope the peroxidase activity is observed in cytoplasmic granules in cells of the convoluted tubules of the ducts. No activity is seen in the acini or in cells of the intralobular striated ducts. The submandibular gland of the rat was negative with respect to peroxidase reaction. The findings are discussed with special reference to the possible correlation between peroxidase activity and iodine metabolism in salivary glands.     

Two ligands signal through the Drosophila PDGF/VEGF receptor to ensure proper salivary gland positioning

The Drosophila embryonic salivary gland is a migrating tissue that undergoes a stereotypic pattern of migration into the embryo. We demonstrate that the migratory path of the salivary gland requires the PDGF/VEGF pathway. The PDGF/VEGF receptor, Pvr, is strongly expressed in the salivary glands, and Pvr mutations cause abnormal ventral curving of the glands, suggesting that Pvr is involved in gland migration. Although the Pvr ligands, Pvf1 and Pvf2, have distinct expression patterns in the Drosophila embryo, mutations for either one of the ligands result in salivary gland migration defects similar to those seen in embryos that lack Pvr. Rescue experiments indicate that the PDGF/VEGF pathway functions autonomously in the salivary gland. The results of this study demonstrate that the Drosophila PDGF/VEGF pathway is essential for proper positioning of the salivary glands.     

Sox9+ cells are required for salivary gland regeneration after radiation damage via the Wnt/β-catenin pathway

Radiotherapy for head and neck cancer can cause serious side effects, including severe damage to the salivary glands, resulting in symptoms such as xerostomia, dental caries, and oral infection. Because of the lack of long-term treatment for the symptoms of xerostomia, current research has focused on finding endogenous stem cells that can differentiate into various cell lineages to replace lost tissues and restore functions. Here, we report that Sox9⁺ cells can differentiate into various salivary epithelial cell lineages under homeostatic conditions. After ablating Sox9⁺ cells, the salivary glands of irradiated mice showed more severe phenotypes and the reduced proliferative capacity. Analysis of online single-cell RNA-sequencing data reveals the enrichment of the Wnt/β-catenin pathway in the Sox9⁺ cell population. Furthermore, treatment with a Wnt/β-catenin inhibitor in irradiated mice inhibits the regenerative capability of Sox9⁺ cells. Finally, we show that Sox9⁺ cells are capable of forming organoids in vitro and that transplanting these organoids into salivary glands after radiation partially restored salivary gland functions. These results suggest that regenerative therapy targeting Sox9⁺ cells is a promising approach to treat radiation-induced salivary gland injury.     

Label-retaining cells in the rat submandibular gland.

To identify stem cells in salivary glands, label-retaining cells (LRCs) were established in rat submandibular glands. Developing and regenerating glands were labeled with bromodeoxyuridine (BrdU). To cause gland regeneration, the glands were injured by duct obstruction. BrdU LRCs were observed in all the parenchymal structures except for the acinus of the glands labeled during regeneration. Among these LRCs, a few, but not many, expressed neither keratin18 (K18; an acinar/duct cell marker) nor alpha-smooth muscle actin (alphaSMA; a myoepithelial cell marker), and thus were putative stem cells. These (K18 and alphaSMA)(neg) LRCs were invariably observed in the intercalated duct and the excretory duct. In the intercalated duct, they were at the proximal end bordering the acinus (the neck of the intercalated duct). Next, to test the above identification, gland extirpation experiments were performed. LRCs were established by labeling developing glands with iododeoxyuridine (IdU) in place of BrdU. Removal of one submandibular gland forced the IdU-LRCs in the remaining gland to divide. They were labeled with chlorodeoxyuridine (CldU). The (K18 and alphaSMA)(neg) LRCs in the neck of the intercalated duct and in the excretory duct did not change in number or in IdU label. The CldU label appeared in these cells and then disappeared. These results indicate that the (K18 and alphaSMA)(neg) LRCs have divided asymmetrically and are thus considered salivary gland stem cells.     

Potential therapeutic effects of induced pluripotent stem cells on induced salivary gland cancer in experimental rats

Salivary gland neoplasms exhibit complex histopathology in a variety of tumor types and treatment options depend largely on the stage of the cancer. Induced pluripotent stem cells (iPS) have been investigated for treating induced salivary gland cancer and for restoring salivary gland function. We investigated iPS treatment for salivary gland cancer both in vitro and in vivo. For our study in vitro, we re-programmed human skin fibroblasts to form iPS cells using a plasmid containing Oct4, Sox2, L-MYC and LIN28. For our study in vivo, we used 30 white male albino rats divided into the following groups of 10: group 1 (control): rats were injected with phosphate-buffered saline (PBS), group 2 induced squamous cell carcinoma (SCC): rat submandibular glands were injected with squamous carcinoma cells (SCC), group 3 (induced SCC/iPS): SCC treated rats treated with 5 × 106 iPS cells. Submandibular glands from rats of all groups were examined histologically and real time PCR was performed for amylase, and COX I and COX II gene expression. We confirmed that submandibular gland specimens included tumor tissue before starting treatment with iPS. iPS treated cases exhibited regeneration of salivary glands, although minor degenerative and vascularization changes remained. The acinar cells regained their proper organization, but continued to exhibit abnormal activity including hyperchromatism. iPS cells may be useful for treating salivary gland carcinomas.     

Proline-rich proteins and glycoproteins: expression of salivary gland multigene families

Our recent research interests have focused on a group of unusual proteins and glycoproteins high in proline content, or the so-called proline-rich proteins (PRPs). The PRPs are tissue-specific expressions of salivary gland multigene families. Normally PRPs are not detected or are present in very low amounts in rat, mouse and hamster salivary glands, but these unusual proteins are dramatically induced by treatment with the catecholamine isoproterenol. The structures and organizations of several PRP mRNAs and PRP genes have been determined. The amino acid sequences of all PRPs show 4 distinct regions, namely, a signal peptide, a transition region, a repeat region and a carboxyl-terminal region. Glycoproteins induced by isoproterenol treatment may be N-glycosylated or O-glycosylated. The N-glycosylated glycoprotein GP-158 from rat submandibular glands has a 12 amino acid glycopeptide which repeats possibly 49 times. Proline-rich proteins of the parotid glands of rats and mice are also greatly induced by dietary tannins. The apparent unique occurrence of PRPs in saliva suggests that one biological role is to neutralize the detrimental effects of dietary tannins and other polyphenols. The upstream regions of the mouse and hamster PRP genes contain cyclic AMP-regulated sequences as demonstrated by deletions and transient transfections. The PRP multigene family members of mouse are all located on chromosome 8.     

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation

ObjectivesSalivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration.Materials and MethodsWe used a duct ligation/deligation‐induced submandibular gland regeneration model of Sprague‐Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin–eosin staining, real‐time PCR (RT‐PCR), immunohistochemistry and Western blotting. We counted the number of Ki67‐positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases.ResultsIntact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67‐positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases‐1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration.ConclusionsOur findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM.     

Characterization and description of a virus causing salivary gland hyperplasia in the housefly,Musca domestica

Abstract. A double-stranded DNA virus was isolated from hyperplasic salivary glands of male and female houseflies, Musca domestica L. (Diptera: Muscidae), collected from a dairy in Alachua County, Florida, U.S.A. Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) of this housefly salivary gland hyperplasia (SGH) virus revealed the presence of two major and eight minor structural polypeptides. Restriction endonuclease analysis indicated that the c. 137 kilobase pair DNA was double-stranded. Weekly sweep-net sampling of the fly population throughout the season (May-October, 1991) showed that 1.5-18.5% of the dissected flies possessed hyperplasic salivary glands. The virus replicated within the nuclei of the salivary gland cells and was transmitted per os to newly-emerged healthy adult flies.     

Effect of rat salivary glands extracts on the proliferation of cultured skin cells - a wound healing model

Objective: Salivary gland secretions play an important role in promotion of wound healing. The healing of intra- or extra-oral wounds is delayed in desalivated rats. However, the specific role of each salivary gland in promoting wound healing is unknown. This study was aimed to investigate the effect of crude extracts of rat salivary glands on a simplified in vitro wound healing model. Design/methods: Cultured human keratinocytes (HaCat) and murine fibroblasts (3T3) were subjected to 48 h serum starvation, and were later activated by extracts of rat salivary glands, 1–10 μg protein/ml of each gland. The resultant cellular metabolic activity of the activated cells was determined 24 h later, measuring reduction of XTT by mitochondrial enzymes, and calculated relatively to positive controls [optimal supplementation of 10% fetal calf serum (FCS)], and negative controls (starved non-supplemented cells). Results: The relative stimulatory effect of parotid (P) extract on the cells was significantly lower than either submandibular (SM) or sublingual (SL) extracts. Under the assumption that physiologically, the cells are exposed to the combined effect of saliva secreted from all the glands, different combinations of the extracts were presented to the cells. The relative stimulation was maximal following treatment with the three glands extracts (P + SM + SL) and exceeded the effect of 10% FCS. Conclusion: The results suggest that each salivary gland has a specific effect on wound healing and the combination of the three extracts has an additive effect but no the sum of all individual glands. This model might be useful to study the wound healing effect of salivary glands. In partial fulfillment of the requirement for MD thesis, The Joyce and Irving Goldman School of Medicine, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.     

The innervation of the salivary gland in the american cockroach: Light and electron microscopic observations

The salivary gland of Periplaneta americana (L.) is innervated from both the stomatogastric nervous system (SNS) and subesophageal ganglion (SEG). Methylene-blue preparations, histological sections and electron microscopy revealed a pair of nerves from the SEG, each of which contains two axons 5–7 μ in diameter, and these are accompanied by several smaller ones. The nerves going to the salivary glands from the SNS contain a dozen or more axons, each less than 2 μ thick. Axons from two sources innervate the efferent salivary ducts, the acini, the anterior ends of the salivary reservoirs, and the reservoir suspensory muscles. A nerve which has reached an acinus forms a plexus upon its surface. Electron micrographs disclose penetration of axons with or without glial wrappings, into the intercellular spaces between gland cells. Axons without glial wrappings have been observed in intimate contact with gland-cell membranes, and several areas which resemble synaptic junctions have been seen.